摘要
目的观察两种方法体外诱导大鼠骨髓间充质干细胞(BMSC)向神经样细胞分化的效果。方法自大鼠股骨中提取骨髓细胞,利用Percoll法进行BMSC分离、纯化及鉴定。取第5代细胞,应用两种方法进行诱导。方法一(BHA组):200μmol/L丁羟基茴香醚(BHA)、10 ng/ml碱性成纤维生长因子(bFGF)和2%二甲基亚砜(DMSO)联合诱导细胞;方法二(RA组):全反式维甲酸(RA)和β-巯基乙醇(β-ME)联合诱导细胞。同时设正常培养的BMSC为对照组。实验过程中对细胞进行形态观察和免疫荧光染色检测神经细胞标志物。结果BHA组诱导5 h,细胞即发生明显的形态改变,出现胞体回缩、突触伸展等神经干细胞的形态特征,而RA组诱导1周未出现神经干细胞形态。免疫荧光染色显示两种方法诱导的细胞均能表达神经前体细胞标志物nestin、β-tubulin和成熟神经元标志物神经元特异性烯醇化酶(NSE),不表达星形胶质细胞标志物胶质原纤维酸性蛋白(GFAP)。BHA组与RA组表达NSE的阳性率分别为80.05%和71.61%,两者相比,差异无统计学意义(P>0.05);对照组不表达NSE,诱导组与对照组比,差异有统计学意义(P<0.05)。结论两种方法均能诱导骨髓间充质干细胞定向分化为表达神经细胞标志物的神经样细胞。
Objective To study the differentiation of rat bone marrow mesenchymal stem cells （BMSCs） into cells with a neural phenotype by two specific treatments in vitro. Methods Isolation, cultivation and amplification of BMSC, and the purified BMSCs of the fifth generation were induced to differentiate into neuron-like cells by using two treatments： （1）treatment with 200 umol/L butylated hydroxyanisole （BHA） in combination with 10 ng/ml bFGF and 2 % DMSO;（2）1umol/L retinoic acid（RA） in combination with 100 umol/L β-ME. Morphological changes of cells during the induction by two treatments were observed under microscopes. The expression of neural markers, nestin, β-tubulin and NSE, of induced cells were showed by immunofluorescence. Results Treatment of BMSCs with BHA for 5 hours led to a dramatic change in morphology, such as cell contraction, neural-like axis production, and the expression of nestin β-tubulin, and characteristic markers for neural precursors and neurons. NSE positive cells were detected for 24 hours. In contrast, cells induced by RA and β-ME didn＇t show typical neural appearance, however, neural markers could be detected 7 days later, indicating a neurogenic differentiation phenotype. Conclusion Both treatments can induce BMSCs differentiating into neuron-like ceils.
出处
《苏州大学学报（医学版）》
CAS
北大核心
2009年第2期197-200,F0002,共5页
Suzhou University Journal of Medical Science
基金
国家自然科学基金资助项目(30671041)
江苏省高校自然科学重大基础研究项目(06KJA18025)
江苏省高校自然科学基金资助一般项目(05KJB310115)
苏州大学医学发展基金资助项目(EE120602)
关键词
骨髓间充质干细胞
诱导分化
神经样细胞
丁羟基茴香醚
全反式维甲酸
bone marrow mesenchymal stem cells
differentiation
neuron-like cells
butylated hydroxyanisole
retinoic acid
